Mastitis is an inflammatory disease of the mammary gland of a mammal.
The inflammation is the result of infection by any of a multitude of bacteria, mycoplasmas, yeast and fungi. The range of different organisms that can cause mastitis and their varying susceptibilities to antibiotics presents the greatest challenge in the treatment and prevention of mastitis in dairy cows.
Bovine mastitis may be caused by Gram negative bacteria such as Escherichia coli, Klebsiella species and Enterbacter species, or by Gram-positive bacteria such as Staphylococcus aureus, Enterococci species, and Streptococci such as Streptococcus uberus, Streptococcus agalactiae and Streptococcus dysgalactiae, and by Mycoplasma bovis. 
Bacterial infection via the teats is the most common cause of mastitis. There is an increased risk of intra-mammary infection during the dry period because the teat is no longer protected by the flow of milk through the teat. Milk is known to have a natural anti-microbial action. Dairy cows are bred for increased milk production. There is a negative correlation between milk production capacity and resistance to mastitis, attributable in part to the increased metabolic stress associated with the synthesis of milk. The large volume of milk may also overwhelm the natural defense systems in the udder.
The teat canal plays a large part in preventing infections in the teat and udder such as mastitis. The teat canal has several anatomical and physiological features that serve as a barrier to penetration by bacterial.
The normal teat canal represents a physical barrier to the penetration of bacteria and is the single most important barrier to udder infection. Essentially, there are three main defense mechanisms offered by the teat canal: adsorptive capacity of keratin for bacteria, desiccation of the canal lumen and desquamation of the keratin lining during milk flow.
Besides being a physical barrier, the canal also contains long chain fatty acids produced by continuous keratinization of epithelial cells that are antibacterial, and a protein called ubiquitin that acts as a general antiseptic against incoming bacteria. In a healthy teat, the keratin plug mechanically traps debris and bacteria and absorb them within the extracellular lipid film (Bramley and Dodd, 1984).
Epithelial desquamation and milk flow during lactation limit bacterial colonization in the teat canal. However, at the beginning of the dry period when milk production ceases, bacteria are able to colonize the teat canal, and multiply and subsequently infect the mammary gland.
Teat seal formulations are currently available—however it is not known for these to include milk components, such as lactoferrin or lactoperoxidase. These form a physical barrier to the teat canal, thereby preventing entry of micro-organisms during the dry period. Some of these formulations include antibiotics, or chemicals known to have anti-microbial activity. Antibiotics require withholding periods which can increase the time and effort required by the farmer, and may not effectively target the pathogens causing the mastitis.
The primary treatment for bovine mastitis is the administration of antibiotics such as penicillin. Antibiotics belonging to the penicillin and cephalosporin family are most effective against Gram-positive bacteria and have poor or strain-dependent activity against Gram-negative pathogens. A study in Poland (Malinowski et al, 2002) found that out of 517 strains of Staphylococcus aureus from subclinical and clinical mastitis, more than 60% were resistant to penicillin and ampicillin, more than 40% were resistant to amoxicillin, 30% were resistant to cephalosporin, 26% were resistant to tetracycline or erythromycin and 54-55% were resistant to lincomycin. Aminoglycosides have a broader spectrum of activity but are not effective against bacteria with rapid growth.
Bovine mastitis is of considerable economic significance to the dairy industry. This is particularly due to the following:                the high cost of the treatment,        the loss of milk during the infected period, and subsequent withholding period following the use of antibiotics. If antibiotics are found in the milk supplied to a dairy company, the whole batch may need to be discarded, and the farmer may face large penalties.        cross-contamination within the herd        long-term loss of milk over the life of the animal due to decreased mammary capacity        
A number of milk components have previously been isolated, purified and discussed in relation to use to prevent or treat mastitis, these include:
Lactoferrin (Lf), a glycoprotein which is present in mammary gland secretion and many other exocrine secretions of mammals, Lf is secreted predominately by surface epithelia into the mucosal environment. Lactoferrin is a multifunctional protein that has antibacterial, antifungal, antiviral, antitumour, anti-inflammatory, and immunoregulatory properties
Lf is produced at high levels in nasal and tracheal passages, and in gastric, genital and opthalmic secretions. Lf is also produced at high levels in neutrophils where it is stored in secondary granules and released during inflammation.
The mechanism by which Lf inhibits microbial growth has not been fully elucidated. Its antimicrobial and anti-inflammatory effects are believed to be as a result of a number of different actions or functions of Lf.
The highly basic N terminal region of bovine lactoferrin is essential for antimicrobial activity. The 25 N-terminal amino acids may be removed by proteases to form Lactoferricin (Lfcin). These proteases may be naturally occurring in milk or serum, and many micro-organisms produce proteases. LFcin is up to a 1000 fold more effective against some micro-organisms than intact lactoferrin. Lfcin has been shown to inhibit a diverse range of microorganisms such as gram-negative bacteria, gram-positive bacteria, yeast, filamentous fungi, and parasitic protozoa, including some antibiotic-resistant pathogens.
Lf binds to lipopolysaccharide. When Gram-negative bacteria are killed by the natural defense system of the animal or by antimicrobial agents the release of lipopolysaccharide from the cell walls of the bacteria provokes an inflammatory response. One of the primary actions of Lf therefore is to bind the LPS and prevent the inflammatory response. Lf also displays an immunomodulatory role by binding with high affinity to bacterial endotoxin, thus protecting against endotoxin lethal shock.
Lf is also an iron binding glycoprotein. Most micro organisms need iron for growth and therefore Lf has the potential to inhibit the growth of bacteria and even kill them by depriving them of iron. The effectiveness of the anti-bacterial activity of Lf depends on the iron requirement of the organism, being availability of exogenous iron, and the concentration and degree of iron saturation of Lf. It has been shown that natural Lf is bacteriostatic against a wide range of micro organisms, including gram negative bacteria with high iron requirements, and some gram positive organisms such as Staphylococcus aureus which is a major mastitis pathogen.
Current commercial applications of bovine Lf include infant formulas, fermented milks, nutritional iron supplements, chewing gums, immune-enhancing nutraceuticals, cosmetic formulas and feed and pet care supplements.
The increased concentrations of endogenous Lf in milk during the dry period, and the bacteriostatic and bactericidal effects of exogenous Lf have lead to research in the use of Lf for treating or preventing mastitis. This research has in the past focused on increasing the purity and extraction rates of Lf to increase the beneficial effect.
Another milk component is Lactoperoxidase (Lp), a protein present in the mammary gland secretion and many other exocrine secretions of mammals.
The Lactoperoxidase system consists of three components—lactoperoxidase, thiocyanate and hydrogen peroxide, which are all present in fresh milk. Lp catalyses the oxidation of thiocyanate by peroxide and generates intermediate products with antibacterial properties. Mammalian cells are not affected by these oxidation products and the Lp system may actually protect cells against the toxic effects of hydrogen peroxide. Thiocyanate is present in the mammary, salivary and thyroid glands and their secretions, in synovial, cerebral, cervical and spinal fluids, in lymph and plasma, and in organs such as stomach and kidney. Hydrogen peroxide, the third component of the Lactoperoxidase system is not normally detected in milk. It may be generated endogenously by polymorphonuclear leucocytes in the process of phagocytosis. Lactobacilli, lactococci and streptococci produce sufficient hydrogen peroxide under aerobic conditions to activate the Lp system.
When provided with the substrate thiocyanate, Lp generates the biocidal compound hypothiocyanite (OSCN−). Halides also act as substrate for Lp. Kussendrager and Hooijdonk (2000) state that oxidation of even small amounts of I− might be significant regarding antimicrobial action because the LP-H2O2—SCN− system is primarily bacteriostatic whereas the LP-H2O2—I− system is bactericidal.
The Lactoperoxidase system has bacteriostatic or bactericidal activity on a variety of susceptible micro-organisms including bacteria, fungi and viruses.
Lp has been used for the preservation of raw milk during storage and transportation, to extend the shelf-life of dairy products, as a preservative in cosmetics and pharmaceuticals. A number of oral hygiene products, such as mouthwashes and toothpaste containing Lp are commercially available. Uses include wound treatment and opthalmic solutions.
Immunoglobulins are an important component of milk and provide passive protection to the suckling young. Although they are not strongly cationic some immunoglobulins, IgG, IgM, IgA and polymeric immunoglobulin receptor (PIGR) are captured by cation exchange. Immunoglobulins are important in the first line of defence against foreign invaders. Immunoglobulins bind to micro-organisms and thus opsonise them so that they are more easily recognized by phagocytic cells.
A number of proteins and peptides belonging to the ribonuclease superfamily have been identified in milk. Some of these have been purified and shown to have antiviral and anti-microbial activity. They are variously described as RNase5, angiogenin 1, angiogenin 2 and lactogenin.
Angiogenin is a small polypeptide that is implicated in the formation of new blood vessels. Angiogenin is unique among the many proteins that are involved in angiogenesis in that it is also an enzyme with an amino acid sequence 33% identical to that of bovine pancreatic ribonuclease (RNase A). Moreover, although Ang has the same general catalytic properties as RNase A—it cleaves preferentially on the 3′ side of pyrimidines and follows a transphosphorylation/hydrolysis mechanism—its activity differs markedly both in magnitude and in specificity.
Although angiogenin contains counterparts for the key catalytic residues of bovine pancreatic RNase A, it cleaves standard RNase substrates 105-106 times less efficiently than does RNase A. Despite this apparent weakness, the enzymatic activity of Ang appears to be essential for biological activity: replacements of important active site residues invariably diminish ribonuclease and angiogenesis activities in parallel, and a substitution that increases enzymatic activity also enhances angiogenic potency (See “Angiogenin” in Wikipedia, May 2006).
The concentration in milk of a number of proteins and peptides increases rapidly in response to trauma or infection. These are known as acute phase proteins (APP) and include lactoferrin, the ribonucleases, N-acetyl glucosaminidase, serum amyloid A, β Defensin and lysozyme.
Work in the dairy field on developing treatment or preventative uses of Lf and Lp have previously been focused towards obtaining more pure and concentrated forms of these proteins from milk.
For example WO 03/002090 discloses the use of Lf, or pharmaceutically acceptable salts thereof to cows to provide a smooth transition of the mammary gland from a lactation period to a dry period. WO 03/002090 discloses the use of small volumes (preferably 100-250 mg/udder) of Lf in ointment or liquid form.
The use of extracted and purified Lf or Lp, or other milk components for the treatment or prevention of mastitis require the extraction, isolation and purification of more concentrated and pure versions of these components.
Unfortunately extraction and purification methods can be time consuming, expensive and hard to develop and implement, especially on a large scale.
Also, the pure products are not fully effective in treating infection.
It is therefore desirable to have available a natural product which is quick and easy to produce which effectively prevents or treats bovine mastitis.
All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in New Zealand or in any other country.
It is acknowledged that the term ‘comprise’ may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term ‘comprise’ shall have an inclusive meaning—i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements. This rationale will also be used when the term ‘comprised’ or ‘comprising’ is used in relation to one or more steps in a method or process.
It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.
Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.